Stefan M. Gehrig

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca2+, which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca2+) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

Cellular mechanisms underlying temporal changes in skeletal muscle protein synthesis and breakdown during chronic β‐adrenoceptor stimulation in mice

Chronic stimulation of β‐adrenoceptors with β‐adrenoceptor agonists (β‐agonists) can induce substantial skeletal muscle hypertrophy, but the mechanisms mediating this muscle growth have yet to be elucidated. We investigated whether chronic β‐adrenoceptor stimulation in mice with the β‐agonist formoterol alters the muscle anabolic response following β‐adrenoceptor stimulation. Twelve‐week‐old C57BL/6 mice were treated for up to 28 days with a once‐daily injection of either saline (control, n= 9) or formoterol (100 μg kg−1; n= 9). Rates of muscle protein synthesis were assessed at either 1, 7 or 28 days of treatment, 6 h after injection. Protein synthesis rates were higher in formoterol‐treated mice at day 7 (∼1.5‐fold, P < 0.05), but not at day 1 or 28. The increased muscle protein synthesis was associated with increased phosphorylation of S6K1 (r= 0.49, P < 0.01). Formoterol treatment acutely reduced maximal calpain activity by ∼25% (P < 0.05) but did not affect atrogin‐1 protein levels and proteasome‐mediated proteolytic activity, despite significantly enhanced phosphorylation of Akt (P < 0.05). Formoterol increased CREB phosphorylation by ∼30% (P < 0.05) and PPARγ coactivator‐1α (PGC‐1α) by 11‐fold (P < 0.05) on day 1 only. These observations identify that formoterol treatment induces muscle anabolism, by reducing calpain activity and by enhancing protein synthesis via increased PI‐3 kinase/Akt signalling.

Intramuscular β2-agonist administration enhances early regeneration and functional repair in rat skeletal muscle after myotoxic injury

Systemic administration of beta(2)-adrenoceptor agonists (beta(2)-agonists) can improve skeletal muscle regeneration after injury. However, therapeutic application of beta(2)-agonists for muscle injury has been limited by detrimental cardiovascular side effects. Intramuscular administration may obviate some of these side effects. To test this hypothesis, the right extensor digitorum longus (EDL) muscle from rats was injected with bupivacaine hydrochloride to cause complete muscle fiber degeneration. Five days after injury, half of the injured muscles received an intramuscular injection of formoterol (100 mug). Muscle function was assessed at 7, 10, and 14 days after injury. A single intramuscular injection of formoterol increased muscle mass and force-producing capacity at day 7 by 17 and 91%, respectively, but this effect was transient because these values were not different from control levels at day 10. A second intramuscular injection of formoterol at day 7 prolonged the increase in muscle mass and force-producing capacity. Importantly, single or multiple intramuscular injections of formoterol did not elicit cardiac hypertrophy. To characterize any potential cardiovascular effects of intramuscular formoterol administration, we instrumented a separate group of rats with indwelling radio telemeters. Following an intramuscular injection of formoterol, heart rate increased by 18%, whereas systolic and diastolic blood pressure decreased by 31 and 44%, respectively. These results indicate that intramuscular injection can enhance functional muscle recovery after injury without causing cardiac hypertrophy. Therefore, if the transient cardiovascular effects associated with intramuscular formoterol administration can be minimized, this form of treatment may have significant therapeutic potential for muscle-wasting conditions.

Insulin‐like growth factor‐I analogue protects muscles of dystrophic mdx mice from contraction‐mediated damage

Contraction‐mediated injury is a major contributing factor to the pathophysiology of muscular dystrophy and therefore therapies that can attenuate this type of injury have clinical relevance. Systemic administration of insulin‐like growth factor‐I (IGF‐I) has been shown to improve muscle function in dystrophic mdx mice, an effect associated with a shift towards a more oxidative muscle phenotype and a reduced susceptibility to contraction‐mediated damage. The actions of IGF‐I in vivo are modulated by IGF binding proteins (IGFBPs), which generally act to inhibit IGF‐I signalling. We tested the hypothesis that an analogue of IGF‐I (LR IGF‐I), which has significantly reduced binding affinity for IGFBPs, would improve the dystrophic pathology by reducing the susceptibility to muscle injury. Dystrophic mdx and wild‐type (C57BL/10) mice were administered LR IGF‐I continuously (∼1.5 mg kg−1 day−1) via osmotic mini‐pump for 4 weeks. Administration of LR IGF‐I reduced the susceptibility of extensor digitorum longus, soleus and diaphragm muscles to contraction damage, as evident from lower force deficits after a protocol of lengthening contractions. In contrast to the mechanism of protection conferred by administration of IGF‐I, the protection conferred by LR IGF‐I was independent of changes in muscle fatigue and oxidative metabolism. This study further indicates that modulation of IGF‐I signalling has therapeutic potential for muscular diseases.

Therapeutic potential of PEGylated insulin-like growth factor I for skeletal muscle disease evaluated in two murine models of muscular dystrophy

OBJECTIVE Duchenne muscular dystrophy (DMD) is a fatal monogenetic disease with affected males displaying severe and progressive muscle wasting and weakness eventually leading to premature death. Possible therapeutic benefits of insulin-like growth factor I (IGF-I) have been studied extensively in various models of muscle disease and DMD with IGF-I as a mediator of improved skeletal muscle regeneration by enhancing myoblast proliferation and differentiation. DESIGN We tested the efficacy of a novel IGF-I analogue, a polyethylene glycol modified IGF-I (PEG-IGF-I), to ameliorate the pathophysiology of muscular dystrophy in two mouse models of DMD. We used mdx mice which lack dystrophin (as in DMD) but exhibit only a relatively mild phenotype, and the dko mouse which is a transgenic model lacking utrophin in addition to dystrophin, and which exhibits a more severe, lethal phenotype like that in DMD. RESULTS In young mdx mice, twice-weekly PEG-IGF-I s.c. injections for 6 weeks protected the diaphragm muscle against fatigue and the tibialis anterior (TA) muscle against contraction-induced injury. However, this beneficial effect of PEG-IGF-I was less pronounced in mdx mice when treatment was initiated later in adulthood. In severely affected dko mice PEG-IGF-I treatment did not affect pathophysiological parameters including animal survival. CONCLUSIONS These data suggest a therapeutic benefit with PEG-IGF-I treatment only in mild muscle pathologies, since its potential to ameliorate the pathophysiology in models of severe muscular dystrophies was limited. Treatment should be initiated only for mild muscle pathologies if functional benefits are to be realised and therefore may be relevant as a short-term therapy to hasten the functional repair of otherwise healthy muscles after injury.

Dysfunctional Muscle and Liver Glycogen Metabolism in mdx Dystrophic Mice

Background Duchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice. Results Dystrophic mdx mice had increased skeletal muscle glycogen (79%, (P<0.01)). Skeletal muscle glycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (P<0.0001). Glycogen synthase activity was 12% higher (P<0.05) but glycogen branching enzyme activity was 70% lower (P<0.01) in mdx compared with wild-type mice. The rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 62% lower activity (P<0.01) in mdx mice resulting from a 24% reduction in PKA activity (P<0.01). In mdx mice glycogen debranching enzyme expression was 50% higher (P<0.001) together with starch-binding domain protein 1 (219% higher; P<0.01). In addition, mdx mice were glucose intolerant (P<0.01) and had 30% less liver glycogen (P<0.05) compared with control mice. Subsequent analysis of the enzymes dysregulated in skeletal muscle glycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; P<0.05) as a possible cause of this phenotype. Conclusion We identified that mdx mice were glucose intolerant, and had increased skeletal muscle glycogen but reduced amounts of liver glycogen.

Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy

What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD.

Emerging drugs for treating skeletal muscle injury and promoting muscle repair

Introduction: Musculoskeletal injuries represent a major global public health problem and muscle injury contributes significantly to the burden of disability and suffering. Drugs that can attenuate muscle trauma and/or hasten muscle repair to restore function can help reduce the economic burden and alleviate personal suffering and financial hardship. Areas covered: This review provides an update on some emerging drugs with therapeutic potential for muscle injury including those that could attenuate damage or improve regeneration. Although there are few (if any) drugs in development specifically for muscle injury, there are numerous drugs in development for cardiovascular complications, such as ischemia-reperfusion, that might also have efficacy for promoting regeneration after similar events in skeletal muscle. Expert opinion: Drugs in development for muscle wasting or inflammatory diseases should also be considered within the context of modulating the events associated with muscle degeneration and regeneration. More rigorous pre-clinical evaluations, especially of a drugs efficacy for improving function, would help minimize false leads and hasten development of effective approaches for treating muscle damage and promoting repair after injury.

Identification of FHL1 as a therapeutic target for Duchenne muscular dystrophy

Utrophin is a potential therapeutic target for the fatal muscle disease, Duchenne muscular dystrophy (DMD). In adult skeletal muscle, utrophin is restricted to the neuromuscular and myotendinous junctions and can compensate for dystrophin loss in mdx mice, a mouse model of DMD, but requires sarcolemmal localization. NFATc1-mediated transcription regulates utrophin expression and the LIM protein, FHL1 which promotes muscle hypertrophy, is a transcriptional activator of NFATc1. By generating mdx/FHL1-transgenic mice, we demonstrate that FHL1 potentiates NFATc1 activation of utrophin to ameliorate the dystrophic pathology. Transgenic FHL1 expression increased sarcolemmal membrane stability, reduced muscle degeneration, decreased inflammation and conferred protection from contraction-induced injury in mdx mice. Significantly, FHL1 expression also reduced progressive muscle degeneration and fibrosis in the diaphragm of aged mdx mice. FHL1 enhanced NFATc1 activation of the utrophin promoter and increased sarcolemmal expression of utrophin in muscles of mdx mice, directing the assembly of a substitute utrophin-glycoprotein complex, and revealing a novel FHL1-NFATc1-utrophin signaling axis that can functionally compensate for dystrophin.

Early functional muscle regeneration after myotoxic injury in mice is unaffected by nNOS absence

Nitric oxide (NO) is an important signaling molecule produced in skeletal muscle primarily via the neuronal subtype of NO synthase (NOS1, or nNOS). While many studies have reported NO production to be important in muscle regeneration, none have examined the contribution of nNOS-derived NO to functional muscle regeneration (i.e., restoration of the muscles ability to produce force) after acute myotoxic injury. In the present study, we tested the hypothesis that genetic deletion of nNOS would impair functional muscle regeneration after myotoxic injury in nNOS(-/-) mice. We found that nNOS(-/-) mice had lower body mass, lower muscle mass, and smaller myofiber cross-sectional area and that their tibialis anterior (TA) muscles produced lower absolute tetanic forces than those of wild-type littermate controls but that normalized or specific force was identical between the strains. In addition, muscles from nNOS(-/-) mice were more resistant to fatigue than those of wild-type littermates (P < 0.05). To determine whether deletion of nNOS affected muscle regeneration, TA muscles from nNOS(-/-) mice and wild-type littermates were injected with the myotoxin notexin to cause complete fiber degeneration, and muscle structure and function were assessed at 7 and 10 days postinjury. Myofiber cross-sectional area was lower in regenerating nNOS(-/-) mice than wild-type controls at 7 and 10 days postinjury; however, contrary to our original hypothesis, no difference in force-producing capacity of the TA muscle was evident between the two groups at either time point. Our findings reveal that nNOS is not essential for functional muscle regeneration after acute myotoxic damage.